WO2017014257A1 - PROTÉINE DE LIAISON À UN ANTICORPS À CAPACITÉ DE LIAISON À UN ANTICORPS DIMINUANT DANS UNE RÉGION DE pH ACIDE - Google Patents

PROTÉINE DE LIAISON À UN ANTICORPS À CAPACITÉ DE LIAISON À UN ANTICORPS DIMINUANT DANS UNE RÉGION DE pH ACIDE Download PDF

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WO2017014257A1
WO2017014257A1 PCT/JP2016/071356 JP2016071356W WO2017014257A1 WO 2017014257 A1 WO2017014257 A1 WO 2017014257A1 JP 2016071356 W JP2016071356 W JP 2016071356W WO 2017014257 A1 WO2017014257 A1 WO 2017014257A1
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protein
amino acid
immunoglobulin
region
domain
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Japanese (ja)
Inventor
正克 西八條
中野　喜之
史憲 鴻池
昌行 高野
吉田　慎一
和信 水口
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Kaneka Corp
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Kaneka Corp
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Priority to EP16827817.4A priority Critical patent/EP3327029A4/fr
Priority to JP2017529918A priority patent/JP6861155B2/ja
Priority to CN201680042996.XA priority patent/CN107849095A/zh
Publication of WO2017014257A1 publication Critical patent/WO2017014257A1/fr
Priority to US15/876,597 priority patent/US20180215795A1/en
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    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
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    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
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    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to an antibody binding protein characterized in that the antibody binding ability in an acidic pH region is reduced.
  • the antibody has a function of specifically binding to a substance called an antigen, and a function of detoxifying and removing a factor having the antigen in cooperation with other biomolecules and cells.
  • the name “antibody” is a name that emphasizes the function of binding to such an antigen, and the substance is called “immunoglobulin (Ig)”.
  • antibody pharmaceuticals using functions of antibodies.
  • antibody drugs work more specifically for target molecules, and are expected to reduce side effects and achieve high therapeutic effects. It contributes to the improvement.
  • the antibody drugs that have been developed are basically monoclonal antibodies, and are produced in large quantities using recombinant cultured cell technology.
  • “Monoclonal antibody” refers to an antibody obtained from a clone derived from a single antibody-producing cell.
  • Most antibody drugs currently on the market are immunoglobulin G (IgG) subclass in terms of molecular structure.
  • Protein A is well known as an immunoglobulin-binding protein having affinity for IgG antibodies.
  • Protein A is one of the cell wall proteins produced by the Gram-positive bacterium Staphylococcus aureus.
  • Non-patent Document 1 Signal sequence S, five immunoglobulin binding domains (E domain, D domain, A domain, B) Domain, C domain) and an XM region which is a cell wall binding domain (Non-patent Document 1).
  • an affinity chromatography column hereinafter referred to as protein A column
  • Patent Document 1 Non-Patent Document 2, Non-Patent Document 3).
  • a typical recombinant protein A is recombinant protein A from which the XM region having no immunoglobulin binding activity has been removed (rProtein A Sepharose (registered trademark), manufactured by GE Healthcare Japan, Inc.). Columns using recombinant protein A from which the XM region has been removed as a ligand have the advantage that non-specific adsorption of proteins can be suppressed compared to conventional products, and are currently widely used industrially.
  • Patent Document 1 recombinant protein A
  • Patent Document 2 recombinant protein A
  • a plurality of Lys is mutated as a ligand.
  • Non-patent Document 1 Non-patent Document 4, Patent Document 3
  • the Z domain is a modified domain in which a mutation that replaces Gly at position 29 with Ala is introduced into the B domain.
  • a mutation that replaces Ala at position 1 of the B domain with Val is also introduced at the same time. This is intended to make it easier to produce a coding gene in which a plurality of domains are linked by genetic engineering. And does not affect the function of the domain (for example, Patent Document 4 includes an example using a mutant in which Val at the 1st position of the Z domain is replaced with Ala).
  • the Z domain is known to have higher alkali resistance than the B domain, and has an advantage in repeated use of the column by washing with an alkaline solution having a high sterilizing and washing effect.
  • ligands imparted with further alkali resistance by substituting Asn with other amino acids have been invented (Patent Documents 5 and 6), and industrial use has also begun.
  • Non-patent Document 5 Another feature of the Z domain is that the ability to bind to the Fab region of immunoglobulin is weakened (Non-patent Document 5).
  • This feature has the advantage that the antibody is easily dissociated in the step of dissociating the bound antibody with an acid (Non-patent Document 1, Patent Document 7). If the antibody is easily dissociated, it is possible to recover a higher concentration of antibody-containing eluate with a smaller volume of eluate.
  • the amount of cell culture solution per batch has exceeded 10,000 liters in antibody drug production, and the antibody expression level has been improved to 10 g / L in the past few years (Non-patent Document 6).
  • the downstream purification process is also inevitably required to cope with an increase in processing scale, and there is a great expectation for technological improvements that enable recovery of higher concentrations of antibody-containing eluate with a small volume of eluate. Big.
  • Patent Document 4 In addition to the Z domain, research based on the C domain of protein A as a modified protein A ligand is underway (Patent Document 4). These ligands are characterized by taking advantage of the high alkali tolerance inherent in the wild-type C domain, and are attracting attention as a new base domain that replaces the Z domain created based on the B domain. . However, as a result of verification with respect to the C domain, there has been a drawback that the antibody is hardly dissociated in the step of dissociating the antibody bound to the C domain with an acid.
  • Non-Patent Document 2 and Patent Document 4 show that the binding ability of the C domain immunoglobulin to the Fab region of the immunoglobulin is strong, and this feature is presumed to be the cause of the antibody being difficult to dissociate with acid. It was. In order to remedy this defect, antibody acid dissociation characteristics were verified using a C domain into which a mutation that replaces Gly at position 29 with Ala was introduced. As a result, the antibody tends to be more easily dissociated than the wild type C domain. Although it was seen, it was still insufficient. It is known that antibodies aggregate at a low pH and antibody activity decreases.
  • the present inventors have compared the activity of many recombinant protein A variants having amino acid substitution mutations. As a result, the hydrophobic amino acid residues at the Fc binding site were changed to other hydrophobic amino acid residues or polar uncharged amino acid residues.
  • the present invention has been completed by finding that the above-mentioned problems can be solved by substituting.
  • the present invention relates to an amino acid sequence derived from the E, D, A, B, or C domain of protein A described in SEQ ID NOs: 1 to 5, wherein the hydrophobic amino acid residue at the Fc binding site is replaced with another hydrophobic amino acid.
  • a protein comprising an amino acid sequence obtained by substitution with a residue or a polar uncharged amino acid residue, wherein the antibody binding ability in an acidic pH region is reduced as compared with that before the substitution About.
  • the hydrophobic amino acid residue of the Fc binding site is preferably Phe corresponding to position 5 of the C domain, Phe corresponding to position 13, Leu corresponding to position 17, or Ile corresponding to position 31.
  • hydrophobic amino acid residues are preferably Gly, Ala, Val, Leu, Ile, Met, Phe, or Trp.
  • the polar uncharged amino acid residue is preferably Ser, Thr, Gln, Asn, Tyr, or Cys.
  • the amino acid corresponding to position 40 of the C domain is preferably an amino acid other than Val.
  • the amino acid sequence is obtained by further substituting a hydrophobic amino acid residue, an acidic amino acid residue, or a polar uncharged amino acid residue with a basic amino acid residue.
  • the present invention also relates to a protein comprising a plurality of domains obtained by linking two or more of the proteins.
  • the present invention also relates to DNA encoding the protein.
  • the present invention also relates to a vector containing the DNA.
  • the present invention also relates to a transformant obtained by transforming a host cell with the vector.
  • the present invention also relates to a cell-free protein synthesis system using the DNA or a method for producing the protein using the transformant.
  • the present invention also relates to an affinity separation matrix obtained by immobilizing the protein as an affinity ligand on a carrier comprising a water-insoluble substrate.
  • the affinity separation matrix preferably binds to a protein containing an immunoglobulin Fc region.
  • the protein containing the Fc region of immunoglobulin is immunoglobulin G or an immunoglobulin G derivative.
  • the present invention also relates to a method for producing the affinity separation matrix, comprising fixing the protein as an affinity ligand to a carrier comprising a water-insoluble substrate.
  • the present invention also relates to a method for purifying a protein containing an immunoglobulin Fc region, comprising adsorbing the protein containing an immunoglobulin Fc region to the affinity separation matrix.
  • the content of the host-derived protein and / or the aggregate of the protein containing the Fc region of immunoglobulin contained in the eluted protein containing the Fc region of immunoglobulin is reduced.
  • the antibody binding ability in the acidic pH region of the affinity separation matrix decreases. Thereby, the antibody can be eluted at a higher pH than before.
  • C-G29A It is a sequence comparison table of the E, D, A, B, and C domains of protein A of Staphylococcus.
  • C-G29A It is a dissolution test result using a 2d affinity separation matrix.
  • Modified C-G29A It is a dissolution test result of Example 8 using 2d affinity separation matrix.
  • the protein of the present invention comprises an amino acid sequence derived from the E, D, A, B, or C domain of protein A described in SEQ ID NOs: 1 to 5, wherein the hydrophobic amino acid residue at the Fc binding site is replaced with another hydrophobic amino acid.
  • a protein comprising an amino acid sequence obtained by substitution with a residue or a polar uncharged amino acid residue, wherein the antibody binding ability in an acidic pH region is reduced as compared with that before the substitution It is.
  • Protein A is a protein consisting of E, D, A, B, and C domains that are immunoglobulin binding domains.
  • the E, D, A, B, and C domains are immunoglobulin binding domains that can bind to regions other than the complementarity determining regions (CDRs) of immunoglobulins, each of which is an immunoglobulin Fc region. , Fab region, and in particular the activity of binding to the Fv region in the Fab region.
  • the origin of protein A is not particularly limited, but protein A derived from staphylococcus is preferable.
  • protein includes any molecule having a polypeptide structure, and a polypeptide chain that is fragmented or linked by peptide bonds is also encompassed by the term “protein”.
  • a “domain” is a unit in a higher-order structure of a protein, which is composed of a sequence of several tens to several hundreds of amino acid residues, and is sufficient for expressing any physicochemical or biochemical function. The unit.
  • the amino acid sequence derived from the domain refers to the amino acid sequence before substitution of amino acids.
  • the amino acid sequence derived from the domain is not limited to the wild-type amino acid sequence of the E, D, A, B, or C domain of protein A, but partially by amino acid substitution, insertion, deletion, and chemical modification. Even a modified amino acid sequence is included in the protein as long as it has a binding ability to the Fc region.
  • Examples of the amino acid sequence derived from the domain include amino acid sequences constituting E, D, A, B, and C domains of protein A of Staphylococcus described in SEQ ID NOs: 1 to 5, and protein A
  • Examples of the E, D, A, B, and C domains are proteins having an amino acid sequence in which a mutation that replaces Gly corresponding to position 29 of the C domain with Ala is introduced.
  • the Z domain in which the mutations A1V and G29A are introduced into the B domain also has an ability to bind to the Fc region, and therefore corresponds to the amino acid sequence derived from the domain.
  • the amino acid sequence derived from the domain is preferably a domain with high chemical stability or a variant thereof.
  • the amino acid sequence derived from the domain has the ability to bind to the Fc region.
  • the sequence identity between the amino acid sequence derived from the domain and the E, D, A, B, or C domain of protein A described in SEQ ID NOS: 1 to 5 is preferably 85% or more, and 90% or more More preferably, it is more preferably 95% or more.
  • the protein of the present invention comprises a hydrophobic amino acid residue at the Fc binding site in the amino acid sequence derived from the E, D, A, B, or C domain of protein A.
  • the amino acid sequence obtained by substituting the group is included.
  • An amino acid substitution means a mutation that deletes the original amino acid and adds another amino acid of a different type at that position.
  • mutation which substitutes an amino acid the wild type or the non-mutated type amino acid is attached
  • G29A a mutation that replaces Gly at position 29 with Ala.
  • hydrophobic amino acid residues in the Fc binding site include Phe corresponding to the 5th position of the C domain, Phe corresponding to the 13th position, Leu corresponding to the 17th position, and Ile corresponding to the 31st position. “Corresponding” means that when the E, D, A, B, and C domains of protein A are aligned as shown in FIG.
  • hydrophobic amino acid residues to be substituted examples include Gly, Ala, Val, Leu, Ile, Met, Phe, and Trp. Among them, Ala, Val, Leu, Ile, and Phe are preferable.
  • the other hydrophobic amino acid residue means a hydrophobic amino acid residue different from the original hydrophobic amino acid residue to be substituted.
  • the original amino acid residue to be substituted is Phe corresponding to the 5th or 13th position of the C domain
  • other hydrophobic amino acid residues examples include the above-mentioned other amino acid residues. Examples include amino acid residues other than Phe.
  • Examples of the polar uncharged amino acid residue to be substituted include Ser, Thr, Gln, Asn, Tyr, and Cys. Among these, Ser, Thr, Gln, Asn, and Tyr are preferable.
  • More specific substitution modes include Phe corresponding to position 5 of the C domain with Ala and Tyr, Phe corresponding to position 13 of the C domain with Tyr, and Leu corresponding to position 17 of the C domain.
  • F5A and F5Y in the C domain, F13Y in the C domain, L17I, L17V and L17T in the C domain, I31L, I31S, I31T and I31V in the C domain, and I31L and I31T in the B domain are preferable.
  • the number of amino acid substitutions at the Fc binding site is not particularly limited as long as the antibody binding ability in the acidic pH region is reduced compared to that before substitution, but in the neutral region and maintenance of the three-dimensional structure of the protein before mutagenesis. From the viewpoint of maintaining the antibody binding ability, 4 or less is preferable, and 2 or less is more preferable. Examples of two amino acid substitutions include substitution of L17I and I31L in the C domain, and substitution of amino acids at positions corresponding to positions 17 and 31 of the C domain in the E, D, A, or B domain in the same manner. The method of doing is mentioned.
  • amino acid substitutions include G29A substitutions in the C domain. Further, in the E, D, A, or B domain, the same substitution of the amino acid at the position corresponding to position 29 of the C domain can be mentioned.
  • arbitrary amino acid substitution includes substitution of an amino acid residue other than Val of Val corresponding to position 40 of the C domain, specifically, substitution of V40Q in the C domain.
  • substitution of a hydrophobic amino acid residue, an acidic amino acid residue, or a polar uncharged amino acid residue with a basic amino acid residue can be mentioned.
  • A12R, L19R, L22R, Q26R in the C domain in the C domain can be mentioned.
  • the hydrophobic amino acid residues at the Fc binding site are replaced with other hydrophobic amino acid residues or polar uncharged
  • the sequence identity between the amino acid sequence obtained by substitution with an amino acid residue and the E, D, A, B, or C domain of protein A described in SEQ ID NOs: 1 to 5 is 85% or more, It is more preferably 90% or more, and further preferably 95% or more.
  • the following amino acid residues are preferably retained 90% or more, more preferably 95% or more; Gln-9, Gln-10, Tyr-14, Pro -20, Asn-21, Leu-22, Gln-26, Arg-27, Phe-30, Leu-34, Pro-38, Ser-39, Leu-45, Leu-51, Asn-52, Gln-55 , And Pro-57 (residue number corresponds to the C domain).
  • the protein of the present invention is characterized in that the antibody binding ability in the acidic pH region is reduced as compared with that before substitution.
  • the acidic pH region include weakly acidic regions, specifically, a range of pH 3-6.
  • Antibody binding ability in the acidic region is determined by pH gradient elution test using IgG Sepharose (Example 1), measurement of antibody binding ability in acidic pH region using intermolecular interaction analyzer, and affinity with immobilized ligand. It can be evaluated by an antibody elution test (Example 5) of the separation matrix. For example, in the case of a pH gradient elution test using IgG Sepharose, a mutant having a reduced antibody binding ability in the acidic region has a higher pH compared to the protein before mutagenesis (for example, C-G29A.2d). Elute.
  • the elution pH of the mutant is preferably 0.05 or higher, more preferably 0.1 or higher.
  • the ligand before mutagenesis eg, C-G29A.2d
  • the antibody recovery rates of the affinity separation matrix to which the antibody is immobilized and the affinity separation matrix to which the mutant is immobilized are compared.
  • the affinity recovery matrix in which the mutant is immobilized preferably has an antibody recovery rate of 1% or more, more preferably 5% or more.
  • the protein of the present invention may be a protein consisting of only a single domain introduced with the amino acid substitution, or a protein consisting of multiple domains obtained by linking two or more domains introduced with the amino acid substitution. There may be.
  • the protein to be linked may be a protein derived from the same type of domain (homopolymer such as homodimer or homotrimer), or a protein derived from different types of domains (heterodimer, Heteropolymers such as heterotrimers).
  • the number of proteins to be linked is preferably 2 or more, more preferably 2 to 10, and even more preferably 2 to 6.
  • monomer proteins are linked to each other and single domains are linked by a method that does not involve a linker amino acid residue, or linked by one or more amino acid residues. Include, but are not limited to, methods.
  • the number of amino acid residues used for linking is not particularly limited, and the linking mode and the number of linking are not particularly limited as long as they do not destabilize the three-dimensional structure of the monomeric protein.
  • the protein of the present invention also includes a fusion protein in which the protein or the protein composed of a plurality of domains is fused as a constituent component with another protein having different functions.
  • fusion proteins include, but are not limited to, proteins in which albumin, GST (glutathione S-transferase), and MBP (maltose binding protein) are fused. By expressing it as a fusion protein with GST and MBP, protein purification can be facilitated.
  • a nucleic acid such as a DNA aptamer, a drug such as an antibiotic, and a polymer such as PEG (polyethylene glycol) are also included in the protein of the present invention as long as they have the same effect as the present invention. .
  • the present invention also relates to DNA encoding the aforementioned protein.
  • the DNA may be any one in which the amino acid sequence obtained by translating the base sequence constituting the DNA constitutes the protein of the present invention.
  • a base sequence can be obtained by using a commonly used known method, for example, a polymerase chain reaction (hereinafter abbreviated as PCR) method. It can also be synthesized by a known chemical synthesis method, and can also be obtained from a DNA library.
  • the base sequence may not be the same as the original base sequence as long as the codon may be substituted with a degenerate codon and it encodes the same amino acid when translated.
  • the DNA of the present invention can be obtained by introducing a site-specific mutation into a conventionally known DNA encoding a wild-type or mutant protein A domain.
  • the introduction of site-specific mutations can be performed using recombinant DNA techniques, PCR methods and the like as follows.
  • the restriction enzyme recognition sequence is introduced. This can be performed by a cassette mutation method in which a portion is cleaved with the restriction enzyme and a region including a site where mutation is desired is removed, and then a DNA fragment mutated only to the target site by chemical synthesis or the like is inserted.
  • site-specific mutation by PCR is, for example, a double double-stranded plasmid in which PCR is performed using a double-stranded plasmid encoding a protein as a template and two kinds of synthetic oligo primers containing mutations complementary to the + and ⁇ strands. This can be done by the primer method.
  • a DNA encoding a protein consisting of a plurality of domains can be prepared by linking a desired number of DNAs encoding the monomer protein (one domain) of the present invention in series.
  • an appropriate restriction enzyme site can be introduced into a DNA sequence, and double-stranded DNA fragmented with a restriction enzyme can be ligated with DNA ligase.
  • a DNA encoding a protein consisting of a plurality of domains can be prepared by applying the above-described mutation introduction method to a DNA encoding protein A (for example, WO 06/004067).
  • a DNA encoding protein A for example, WO 06/004067.
  • the base sequences encoding each monomer protein are the same, homologous recombination may be induced in the host.
  • the sequence identity between the base sequences of DNA encoding the monomeric protein is preferably 90% or less, and more preferably 85% or less.
  • the vector of the present invention comprises a base sequence encoding the aforementioned protein or a protein consisting of a plurality of domains, and a promoter that can function in a host operably linked to the base sequence. Usually, it can be obtained by ligating or inserting a DNA encoding the aforementioned protein into a vector.
  • the vector for inserting the gene is not particularly limited as long as it can replicate autonomously in the host, and plasmid DNA or phage DNA can be used as the vector.
  • vectors for inserting genes include pQE vectors (Qiagen), pET vectors (Merck), and pGEX vectors (GE Healthcare Japan ( Vector) and the like.
  • pUB110 known as a Bacillus subtilis vector, pHY500 (JP-A-2-31682), pNY700 (JP-A-4-278091), pNU211R2L5 (special) (Kaihei 7-170984), pHT210 (JP-A-6-133782), or pNCMO2 (JP-A 2002-238569), which is a shuttle vector between Escherichia coli and Brevibacillus bacteria, can be used. .
  • a transformant can be obtained by transforming a host with a vector.
  • the host is not particularly limited, but for mass production at low cost, Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus, Corynebacterium genus Bacteria (eubacteria) such as (Corynebacterium) can be preferably used. More preferably, Gram-positive bacteria such as Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus and Corynebacterium genus are preferable. More preferably, a bacterium belonging to the genus Brevibacillus, for which an example of application to mass production of protein A (WO 06/004067) is known, is preferred.
  • Brevibacillus genus bacteria are not particularly limited, and examples thereof include Brevibacillus luagri, B. et al. borstelensis, B.M. brevis, B.M. centrosporus, B.M. choshinensis, B. et al. formusus, B.M. invocatus, B.M. laterosporus, B.I. limnophilus, B. et al. parabrevis, B.I. reuszeri, B.M. thermorubber.
  • Brevibacillus brevis 47 strain JCM6285
  • Brevibacillus brevis 47K strain (FERM BP-2308)
  • Brevibacillus brevis 47-5Q strain (JCM8970)
  • Brevibacillus choshinensis HPD31 strain (FERM BP-1087)
  • Brevibacillus choshinensis HPD31-OK strain (FERM BP-4573).
  • a mutant strain (or derivative strain) such as a protease-deficient strain, a high-expressing strain, or a spore-forming-deficient strain of the aforementioned Brevibacillus bacterium may be used depending on the purpose such as improvement of the production amount. .
  • Brevibacillus choshinensis HPD31-derived Brevibacillus choshinensis HPD31-OK JP-A-6-296485), which is a protease mutant derived from Brevibacillus choshinensis HPD31, and Brevibacillus choshinensis HPD31, which does not have spore-forming ability.
  • -SP3 International Publication No. 05/045005
  • Examples of methods for introducing a vector into a host cell include a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, an Agrobacterium infection method, a particle gun method, or a polyethylene glycol method. However, it is not limited to these.
  • examples of a method for expressing the function of the obtained gene in a host include a method for incorporating the gene obtained in the present invention into a genome (chromosome).
  • a protein can be produced by the above-described transformant or a cell-free protein synthesis system using DNA.
  • the transformed cell When a protein is produced using a transformant, the transformed cell is cultured in a medium, and is cultured in a culture cell (including the cell periplasm region) or in a culture solution (outside the cell). It can be produced by producing and accumulating proteins, and a desired protein can be collected from the culture.
  • the protein When a protein is produced using a transformed cell, the protein can be accumulated in the cell and / or in the periplasmic region of the transformant. In this case, accumulation in the cell is advantageous in that it prevents oxidation of the expressed protein and there is no side reaction with the medium components, and accumulation in the periplasmic region can suppress degradation by intracellular protease. This is advantageous.
  • it is also possible to secrete the protein outside the transformant. In this case, the cell disruption and extraction steps are unnecessary, which is advantageous in that the manufacturing cost can be reduced.
  • the method of culturing the transformed cell of the present invention in a medium is performed according to a usual method used for host culture.
  • the medium used for culturing the obtained transformant is not particularly limited as long as the protein can be produced with high efficiency and high yield.
  • carbon sources and nitrogen sources such as glucose, sucrose, glycerol, polypeptone, meat extract, yeast extract, and casamino acid can be used.
  • inorganic salts such as potassium salt, sodium salt, phosphate, magnesium salt, manganese salt, zinc salt, iron salt and the like are added as necessary.
  • an auxotrophic host cell a nutrient substance required for growth may be added. If necessary, antibiotics such as penicillin, erythromycin, chloramphenicol, neomycin may be added.
  • protease inhibitors ie, phenylmethanesulfonylfluoride (PMSF), benzamideline, 4- (2- Aminoethyl) -benzensulfonyl fluoride (AEBSF), Antipain, Chymostatin, Leupeptin, Pepstatin A, Phosphoramidon, Aprotinin, Ethylenedietamine Also good.
  • molecular chaperones such as GroEL / ES, Hsp70 / DnaK, Hsp90, Hsp104 / ClpB may be used.
  • it can be made to coexist with the protein of the present invention by a technique such as coexpression or fusion proteinization.
  • there are techniques such as adding an additive that promotes correct folding to the medium, and culturing at a low temperature. is not.
  • LB medium tryptone 1%, yeast extract 0.5%, NaCl 1%
  • 2 ⁇ YT medium tryptone 1.6%, yeast Extract 1.0%, NaCl 0.5%) and the like.
  • TM medium peptone 1%, meat extract 0.5%, yeast extract 0.2%, glucose 1%, pH 7.0
  • 2SL medium peptone 4%, yeast extract 0.5%, glucose 2%, pH 7.2
  • the culture temperature is 15 to 42 ° C., preferably 20 to 37 ° C.
  • the protein of the present invention is cultured in the cultured cells (in the periplasm region) by aerobically culturing for several hours to several days under aeration and stirring conditions. Or accumulated in a culture solution (extracellular) and collected. In some cases, the culture may be performed anaerobically by blocking aeration.
  • the recombinant protein produced by separating the cultured cells and the supernatant containing the secreted protein by a general separation method such as centrifugation or filtration after the completion of the culture. can be recovered.
  • the cells when accumulated in cultured cells (including in the periplasm region), for example, the cells are collected from the culture solution by a method such as centrifugation or filtration, and then the cells are sonicated.
  • the protein accumulated and produced in the cells can be recovered by crushing by a French press method and / or solubilizing by adding a surfactant or the like.
  • the cell-free protein synthesis system is not particularly limited.
  • a prokaryotic cell-derived, plant cell-derived, higher animal cell-derived synthesis system or the like is used. Can do.
  • the protein of the present invention can be purified by affinity chromatography, cation or anion exchange chromatography, gel filtration chromatography or the like alone or in appropriate combination.
  • Confirmation that the obtained purified substance is the target protein can be performed by usual methods such as SDS polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis, Western blotting and the like.
  • the protein produced by the above method can be immobilized as an affinity ligand on a carrier composed of a water-insoluble base material to produce an affinity separation matrix.
  • affinity ligand is a substance that selectively collects (binds) a target molecule from a set of molecules based on the affinity between specific molecules represented by the binding of an antigen and an antibody. It is a term indicating (functional group), and in the present invention, it refers to a protein that specifically binds to immunoglobulin.
  • the expression “ligand” is also synonymous with “affinity ligand”.
  • Examples of the carrier composed of a water-insoluble substrate used in the present invention include inorganic carriers such as glass beads and silica gel, crosslinked polymers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, crystalline cellulose, crosslinked Examples thereof include organic carriers composed of polysaccharides such as cellulose, crosslinked agarose and crosslinked dextran, and organic-organic, organic-inorganic and other composite carriers obtained by a combination thereof.
  • GCL2000 which is a porous cellulose gel
  • Sephacryl® S-1000 in which allyldextran and methylenebisacrylamide are covalently crosslinked
  • Toyopearl which is a methacrylate-based carrier
  • Sepharose® CL4B which is an agarose-based crosslinked carrier
  • Cellufine which is a cellulosic crosslinking carrier.
  • the water-insoluble carrier in the present invention is not limited to these exemplified carriers.
  • the water-insoluble carrier used in the present invention desirably has a large surface area in view of the purpose and method of use of the affinity separation matrix, and is preferably a porous material having a large number of pores of an appropriate size.
  • the form of the carrier can be any of beads, monoliths, fibers, membranes (including hollow fibers), and any form can be selected.
  • the ligand may be bound to the carrier by a conventional coupling method using an amino group, a carboxyl group, or a thiol group present in the ligand.
  • the support is activated by reacting the support with cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine, sodium periodate, or the like (or on the support surface).
  • introducing a reactive functional group a method of immobilizing by performing a coupling reaction with a compound to be immobilized as a ligand, a condensation reagent such as carbodiimide in a system in which a compound to be immobilized as a carrier and a ligand exists, or
  • the immobilization method include addition of a reagent having a plurality of functional groups in the molecule such as glutaraldehyde, condensation, and crosslinking.
  • a spacer molecule composed of a plurality of atoms may be introduced between the ligand and the carrier, or the ligand may be directly immobilized on the carrier. Therefore, for the immobilization, the protein of the present invention may be chemically modified, or an amino acid residue useful for immobilization may be added.
  • amino acids useful for immobilization include amino acids having functional groups useful for immobilization chemical reactions in the side chain, such as Lys containing an amino group in the side chain, and thiol groups in the side chain. Cys containing is mentioned.
  • the essence of the present invention is that the effect imparted to the protein in the present invention is similarly imparted to the matrix in which the protein is immobilized as a ligand, and no matter how it is modified or altered for immobilization. And within the scope of the present invention.
  • the affinity separation matrix is obtained by immobilizing the protein of the present invention, the affinity separation matrix can be bound to a protein containing the Fc region of immunoglobulin based on the activity of the protein of the present invention itself. Therefore, the protein containing the Fc region of immunoglobulin can be separated and purified by affinity column chromatography purification method using the protein of the present invention and the affinity separation matrix.
  • a protein containing an Fc region of an immunoglobulin refers to a protein containing a site on the Fc region side to which protein A binds. However, if protein A can be bound, it does not have to be a protein that completely contains the Fc region.
  • immunoglobulin G examples include, but are not limited to, immunoglobulin G or an immunoglobulin G derivative.
  • immunoglobulin G derivative means, for example, a chimeric immunoglobulin G in which a part of the domain of human immunoglobulin G is replaced with a domain of immunoglobulin G of another species and a CDR of human immunoglobulin G
  • the (Complementarity Determinig Regions) part is replaced with the CDR part of an antibody of another species, and the humanized immunoglobulin G is fused, the immunoglobulin G obtained by molecular modification of the sugar chain of the Fc region, the Fv region of the human immunoglobulin G, and It is a generic name for modified artificial proteins to which protein A can bind, such as artificial immunoglobulin G fused with Fc region, and fusion protein fused with useful protein and Fc region of human immunoglobulin G.
  • Useful proteins include various receptors, cytokines, hormones, and enzymes.
  • the receptor includes the extracellular region of TNF receptor, VEGF receptor, CTLA4, the cytokine includes the thrombopoietin receptor peptide, the hormone includes the GLP-1 peptide, and the enzyme includes blood coagulation.
  • the enzyme includes blood coagulation. Examples include Factor VIII, blood coagulation factor IX, and phosphatase.
  • the binding region is broadly defined as Fab region (particularly Fv region) and Fc region, since the three-dimensional structure of the antibody is already known, the object to which the protein of the present invention and the affinity separation matrix bind.
  • the protein to be obtained may be a protein in which the Fab region or the Fc region is further modified (fragmentation, etc.) while retaining the three-dimensional structure of the region to which protein A binds in terms of protein engineering.
  • the protein containing the immunoglobulin Fc region is contacted with the affinity separation matrix containing the ligand immobilized on the carrier to thereby affinity the protein containing the immunoglobulin Fc region.
  • Adsorb to a separation matrix Specifically, after the buffer containing the protein containing the Fc region of immunoglobulin is adjusted to be neutral, the solution is passed through an affinity column packed with an affinity separation matrix, and the immunoglobulin Fc region is passed through. Adsorb protein containing.
  • buffer examples include citric acid, 2- (N-morpholino) ethansulfonic acid (MES), Bis-Tris, N- (2-Acetamido) iminodiacetic acid (ADA), Piperazine-1,4-bis (2- etheresulfonic acid) (PIPES), N- (2-Acetamido) -2-aminoethanesulfonic acid (ACES), 3- (N-Morpholino) -2-hydroxypropionic acid (MOPSO), N, N-Brys-hixy 2-aminoethanesulfonic acid (BES), 3- (N-morpholino) prop nesulonic acid (MOPS), N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 4- (2-hydroxyethylethyl) -1-piperazine etheric acid (HEPES), 3-thieol (HEPESol) 1-piperazinyl] propanesulfonic acid (EPPS), Tri
  • the pH at which the protein containing the Fc region of immunoglobulin is adsorbed to the affinity separation matrix is preferably pH 6.5 to 8.5, more preferably pH 7 to 8.
  • the temperature at which the antibody-like protein is adsorbed to the affinity separation matrix is preferably 1 to 40 ° C, and more preferably 4 to 25 ° C.
  • an appropriate amount of pure buffer may be passed through the affinity column to wash the interior of the column.
  • the desired antibody-like protein is adsorbed to the affinity separation matrix in the column.
  • the same buffer as that used in the first step can be used.
  • an eluate having a pH of 3.5 or more is brought into contact with an affinity separation matrix to elute the protein containing the immunoglobulin Fc region.
  • the eluate include citrate buffer, acetate buffer, phosphate buffer, glycine buffer, formate buffer, propionate buffer, ⁇ -aminobutyric acid buffer, and lactic acid buffer.
  • the pH of the eluate is pH 3.0 or higher, it is possible to recover the antibody.
  • using an eluate having a higher pH is preferable because it can avoid antibody aggregation and activity reduction.
  • pH 3.5 or higher is more preferable, pH 3.6 or higher is further preferable, pH 3.75 or higher is even more preferable, pH 3.8 or higher is particularly preferable, pH 3.9 or higher is most preferable, and pH 4.0 or higher.
  • the upper limit of the pH of the eluate is preferably pH 6.0.
  • the affinity separation matrix of the present invention can elute antibodies particularly under high pH conditions, it is preferable that a part of the gradient elution contains pH 4-6 eluate.
  • a surfactant for example, Tween 20 or Triton-X100
  • a chaotropic agent for example, urea or guanidine
  • an amino acid for example, arginine
  • the pH in an affinity column packed with an affinity separation matrix when eluting a protein containing the Fc region of an immunoglobulin is preferably pH 3.0 or more, more preferably pH 3.5 or more.
  • PH 3.6 or more is more preferable, pH 3.75 or more is still more preferable, pH 3.8 or more is particularly preferable, pH 3.9 or more is most preferable, pH 4.0 or more More preferably, Elution at pH 3.0 or higher can reduce antibody damage (Gose S. et al., Biotechnology and bioengineering, 2005, Vol. 92, No. 6).
  • the upper limit of the pH in the affinity column packed with the affinity separation matrix when eluting the protein containing the Fc region of immunoglobulin is preferably pH 6.0.
  • a protein containing an immunoglobulin Fc region can be dissociated under more acidic acidic elution conditions, and an elution peak profile when a protein containing an immunoglobulin Fc region is eluted under acidic conditions Is sharper.
  • the sharper chromatographic elution peak profile enables a higher concentration of antibody-containing eluate to be collected with a smaller volume of eluate.
  • the temperature at which the protein containing the Fc region of immunoglobulin is eluted is preferably 1 to 40 ° C, more preferably 4 to 25 ° C.
  • the recovery rate of the protein containing the Fc region of the immunoglobulin recovered by the purification method of the present invention is preferably 90% or more, and more preferably 95% or more.
  • the recovery rate is calculated by the following formula, for example.
  • Recovery rate (%) (concentration of protein containing Fc region of eluted immunoglobulin (mg / mL) ⁇ elution volume (ml)) ⁇ (concentration of protein containing Fc region of loaded immunoglobulin (mg / ml) mL) x amount of liquid loaded (ml)) x 100
  • contamination of a host-derived protein (HCP) for expressing a protein containing the Fc region of an immunoglobulin can be reduced. Further, contamination of protein aggregates containing the Fc region of immunoglobulin can be reduced. Contamination of these proteins may increase the load of the purification process in antibody production (increase in man-hours and decrease in yield) and impurity proteins may cause serious side effects as pharmaceuticals. The problem can be avoided.
  • HCP host-derived protein
  • the affinity separation matrix of the present invention is effective for separating a protein containing an Fc region of an immunoglobulin and a host-derived protein.
  • the host cell from which the host cell protein is derived is a cell capable of expressing a protein containing the Fc region of an immunoglobulin, and examples thereof include CHO cells and Escherichia coli for which genetic recombination techniques have been established.
  • These host-derived proteins can be quantified by a commercially available immunoassay kit. For example, if a CHO HCP ELISA kit (Cygnus) is used, proteins derived from CHO cells can be quantified.
  • the affinity separation matrix of the present invention at least 1%, 5%, or 10% of the aggregate of proteins containing the Fc region of immunoglobulin, for example, the total amount of protein containing the Fc region of immunoglobulin in the eluate It is effective in purifying a protein containing the Fc region of non-aggregated immunoglobulin from a solution containing the aggregate of the above-mentioned aggregate and removing the aggregate.
  • the content of the aggregate can be analyzed and quantified by, for example, gel filtration chromatography.
  • the affinity separation matrix of the present invention contains an appropriate strong acid or strong alkaline pure buffer (appropriate denaturing agent or organic solvent) that does not completely impair the function of the ligand compound or the carrier substrate. It may be reused by passing it through and washing it.
  • the affinity of the protein of the present invention and the affinity separation matrix to the protein containing the Fc region of immunoglobulin is measured by, for example, a biosensor such as a Biacore system (manufactured by GE Healthcare Japan Co., Ltd.) using the surface plasmon resonance principle. Can be tested.
  • the affinity of the protein of the present invention for the immunoglobulin may be such that the binding constant (K A ) is 10 6 (M ⁇ 1 ) or more when the affinity for the human immunoglobulin G preparation is measured by the Biacore system described below. Preferably, it is more preferably 10 7 (M ⁇ 1 ) or more.
  • the measurement conditions may be any conditions as long as the binding signal can be detected when the protein of the present invention binds to the Fc region of an immunoglobulin, and neutral conditions at a temperature of 20 to 40 ° C. (constant temperature) and a pH of 6 to 8. It can be easily evaluated by measuring at.
  • binding immunoglobulin molecule examples include a polyclonal antibody, gamma globulin Nichiyaku (human immunoglobulin G) (manufactured by Nippon Pharmaceutical Co., Ltd.) and a commercially available monoclonal antibody.
  • the difference in affinity is obtained by obtaining the binding reaction curve for the same immunoglobulin molecule under the same measurement conditions, and the protein before introducing the mutation and the protein after introducing the mutation using the binding parameters obtained when analyzed. Can be easily verified by those skilled in the art.
  • binding parameter for example, a binding constant (K A ) or a dissociation constant (K D ) can be used (Nagata et al., “Real-time analysis experiment method of biological substance interaction”, Springer Fairlark Tokyo, 1998. 41).
  • the binding constant between Fab of each domain of the present invention and Fab is determined by immobilizing an immunoglobulin Fab fragment belonging to the VH3 subfamily on a sensor chip using a Biacore system, at a temperature of 25 ° C. and pH 7.4.
  • each domain variant can be determined in an experimental system in which a flow path is added.
  • the binding constant is sometimes referred to as an affinity constant, but the definition of both is basically the same.
  • Various proteins obtained in the examples are expressed in the form of “alphabet showing domain-introduced mutation (Wild in wild type)”.
  • the wild-type C domain of protein A is referred to as “C-wild”
  • the C domain mutant into which mutation G29E has been introduced is referred to as “C-G29E”.
  • the notation of the mutant in which two kinds of mutations are introduced at the same time is written together using a slash.
  • the mutation G29E and the C domain mutant into which the mutation S13L is introduced are referred to as “C-G29E / S13L”.
  • a protein in which a plurality of single domains are linked is expressed by adding “d” to the linked number after a period.
  • a protein in which 5 mutations of a C domain mutant introduced with mutation G29E and mutation S13L are represented as “C-G29E / S13L.5d”.
  • Example 1 Evaluation of antibody binding ability of C domain mutant using IgG-immobilized carrier C-G29A.
  • Modified type in which the amino acid substitution mutation described in Table 1 is introduced into DNA (SEQ ID NO: 7) having a PstI recognition site at the 5 ′ end and an XbaI recognition site at the 3 ′ end of DNA encoding 2d (SEQ ID NO: 6) C-G29A.
  • the 2d artificially synthesized gene was totally synthesized by outsourcing (manufactured by Eurofin Genomics).
  • the expression plasmid after this subcloning is digested with restriction enzymes PstI and XbaI (Takara Bio), and the obtained DNA fragment is ligated to the Brevibacillus expression vector pNCMO2 (Takara Bio) digested with the same restriction enzymes. Modified C-G29A.
  • An expression plasmid was prepared in which a DNA encoding the 2d amino acid sequence was inserted into the Brevibacillus expression vector pNCMO2.
  • Escherichia coli JM109 strain was used for the preparation of the plasmid.
  • Brevibacillus choshinensis SP3 strain (manufactured by Takara Bio Inc.) was transformed with the obtained plasmid, and modified C-G29A.
  • a gene recombinant that secreted and produced 2d was bred.
  • the cells were subjected to shaking culture at 30 ° C. for 3 days in manganese 0.001% and zinc chloride 0.0001%.
  • the cells were removed from the culture by centrifugation (15,000 rpm, 25 ° C., 5 minutes), and then modified C-G29A. The concentration of 2d was measured. Using an IgG-immobilized carrier, modified C-G29A. 2d and C-G29A. A 2d dissolution test was performed under the following conditions.
  • Carrier IgG Sheparose FF (manufactured by GE Healthcare) Column: Omnifit column (manufactured by Diva Industries), column diameter 0.66 cm, bed height 6.4 cm, column volume: 2.19 mL Flow rate: 0.8 mL / min, contact time 2.7 min Load amount: 470 ⁇ L (ligand concentration 1.3 mg / mL) Equilibrated buffer: 50 mM Tris-HCl, 150 mM NaCl buffer pH 7.5 Elution conditions: 50 mM citrate buffer pH 6.0 ⁇ 50 mM citrate buffer pH 3.0 (20 CV)
  • C-G29A Modified C-G29A.2 based on 2d elution pH. The difference in elution pH of 2d was calculated. The results are shown in Table 1. Any modified C-G29A. 2d is also C-G29A. Compared to 2d, the elution pH from the IgG-immobilized carrier was higher. This result is shown in C-G29A. Compared to the carrier on which 2d is immobilized, modified C-G29A. It is shown that the carrier on which 2d is immobilized can elute the antibody at a higher pH.
  • Example 2 Evaluation of antibody binding ability of C domain mutant using intermolecular interaction analyzer Obtained in Example 1 using biosensor Biacore 3000 (manufactured by GE Healthcare) using surface plasmon resonance The affinity of each of the various proteins with immunoglobulins was analyzed.
  • a human immunoglobulin G preparation hereinafter referred to as human IgG
  • human IgG human immunoglobulin G preparation
  • Human IgG was immobilized on a sensor chip, and various proteins were allowed to flow on the chip to detect their interaction. Immobilization of human IgG on the sensor chip CM5 is carried out by an amine coupling method using N-hydroxysuccinimide (NHS) and N-ethyl-N ′-(3-dimethylaminopropyl) carbohydrate hydride (EDC). Ethanolamine was used for the sensor chip (sensor chips and immobilization reagents were all manufactured by GE Healthcare).
  • NHS N-hydroxysuccinimide
  • EDC N-ethyl-N ′-(3-dimethylaminopropyl) carbohydrate hydride
  • the human IgG solution is prepared by dissolving gamma globulin “Nichiyaku” (manufactured by Nippon Pharmaceutical Co., Ltd.) in a standard buffer (20 mM NaH 2 PO 4 -Na 2 HPO 4 , 150 mM NaCl, pH 7.4) at 1.0 mg / mL. Prepared.
  • the human IgG solution was diluted 100 times with an immobilization buffer (10 mM CH 3 COOH—CH 3 COONa, pH 5.0), and the human IgG was immobilized on the sensor chip according to the protocol attached to the Biacore 3000.
  • a reference cell serving as a negative control was prepared by performing a process of fixing ethanolamine after activation by EDC / NHS for another flow cell on the chip.
  • Various proteins are appropriately prepared in the range of 10 to 1000 nM using a running buffer (20 mM NaH 2 PO 4 -Na 2 HPO 4 , 150 mM NaCl, 0.005% P-20, pH 7.4). 3 types of solutions having different protein concentrations were prepared), and each protein solution was added to the sensor chip for 30 seconds at a flow rate of 20 ⁇ L / min. At a measurement temperature of 25 ° C., a binding reaction curve at the time of addition (binding phase, 30 seconds) and after completion of the addition (dissociation phase, 60 seconds) was observed in order.
  • modified C-G29A As shown in Table 2, modified C-G29A.
  • the binding parameters for 2d human IgG are C-G29A. Similar to 2d (control). Specifically, the binding constant for human IgG was 10 8 M ⁇ 1 or more for all ligands. Any modified C-G29A. In the neutral pH region 2d, C-G29A. The antibody binding ability was comparable to that of 2d.
  • Example 3 Evaluation of antibody binding ability of B domain mutant using IgG-immobilized carrier B-G29A.
  • Modified B in which the amino acid substitution mutation described in Table 3 is introduced into DNA (SEQ ID NO: 9) having a PstI recognition site at the 5 ′ end and an XbaI recognition site at the 3 ′ end of DNA encoding 2d (SEQ ID NO: 8) -G29A.
  • the 2d artificially synthesized gene was totally synthesized by outsourcing (manufactured by Eurofin Genomics).
  • recombinant expression was carried out, and the resulting culture supernatant was subjected to an elution test using an IgG-immobilized carrier.
  • Example 4 Evaluation of antibody binding ability of B domain mutant using intermolecular interaction analyzer The affinity of various proteins obtained in Example 3 with immunoglobulin was analyzed in the same manner as in Example 2. The results are shown in Table 4.
  • Modified B-G29A The binding parameters for 2d human IgG are B-G29A. Similar to 2d (control). Any modified B-G29A. In the neutral pH region, 2G before B. G29A. The antibody binding ability was comparable to that of 2d.
  • Example 5 Modified C-G29A. 2d affinity separation matrix antibody elution test
  • the culture obtained by culturing in the same manner as in Example 1 was centrifuged to separate the cells, and the pH was adjusted to 4.5 by adding acetic acid to the culture supernatant. And allowed to stand for 1 hour to precipitate the protein of interest.
  • the precipitate was collected by centrifugation and dissolved in a buffer solution (50 mM Tris-HCl, pH 8.5).
  • a buffer solution 50 mM Tris-HCl, pH 8.5
  • the target protein was purified by anion exchange chromatography using a HiTrap Q column (GE Healthcare Bioscience Co., Ltd.).
  • the target protein solution is added to a HiTrap Q column equilibrated with anion exchange buffer A (50 mM Tris-HCl, pH 8.0), washed with anion exchange buffer A, The target protein eluted in the middle was fractionated with a salt concentration gradient using anion exchange buffer A and anion exchange buffer B (50 mM Tris-HCl, 1M NaCl, pH 8.0).
  • anion exchange buffer A 50 mM Tris-HCl, 1M NaCl, pH 8.0
  • anion exchange buffer B 50 mM Tris-HCl, 1M NaCl, pH 8.0
  • the collected target protein solution was dialyzed against ultrapure water, and the aqueous solution after dialysis was used as a final purified sample.
  • all the protein purification by the chromatography using a column was implemented using the AKTA york system (made by GE Healthcare Bioscience Co., Ltd.).
  • a water-insoluble substrate 1 mL of a commercially available activated prepack column “Hitrap NHS activated HP” (manufactured by GE Healthcare) was used. This column is based on cross-linked agarose and has been introduced with N-hydroxysuccinimide (NHS) groups for immobilizing proteinaceous ligands. According to the product manual, the final purified sample was immobilized as a ligand, and an affinity separation matrix was prepared.
  • Hitrap NHS activated HP commercially available activated prepack column “Hitrap NHS activated HP” (manufactured by GE Healthcare) was used. This column is based on cross-linked agarose and has been introduced with N-hydroxysuccinimide (NHS) groups for immobilizing proteinaceous ligands. According to the product manual, the final purified sample was immobilized as a ligand, and an affinity separation matrix was prepared.
  • NHS N-hydroxysuccinimide
  • a solution obtained by diluting the final purified sample with a coupling buffer (0.2 M sodium carbonate, 0.5 M NaCl, pH 8.3) to a final concentration of about 13 mg / mL was prepared.
  • 1 mL of the sample diluted solution prepared above was added at the same flow rate, and the obtained protein was immobilized on the column by plugging the top and bottom of the column and allowing to stand at 25 ° C. for 30 minutes.
  • Example 6 Evaluation of antibody-binding ability of C domain mutant using IgG-immobilized carrier C-G29A.
  • the 2d artificially synthesized gene was totally synthesized by outsourcing (manufactured by Eurofin Genomics).
  • Example 2 After preparing a plasmid in the same manner as in Example 1, the gene recombinant was bred, and modified C-G29A. A culture solution containing 2d was obtained. Using an IgG-immobilized carrier, modified C-G29A. 2d and C-G29A. The 2d dissolution test was performed under the same conditions as in Example 1.
  • C-G29A Modified C-G29A.2 based on 2d elution pH. The difference in elution pH of 2d was calculated. The results are shown in Table 6. Any modified C-G29A. 2d is also C-G29A. Compared to 2d, the elution pH from the IgG-immobilized carrier was higher. This result is shown in C-G29A. Compared to the carrier on which 2d is immobilized, modified C-G29A. It is shown that the carrier on which 2d is immobilized can elute the antibody at a higher pH.
  • Example 7 Evaluation of antibody binding ability of C domain mutant using intermolecular interaction analyzer Obtained in Example 6 using biosensor Biacore 3000 (manufactured by GE Healthcare) using surface plasmon resonance The various proteins were analyzed for affinity with immunoglobulin under the same conditions as in Example 2. Human IgG was used as the immunoglobulin.
  • modified C-G29A As shown in Table 7, modified C-G29A.
  • the binding parameters for 2d human IgG are C-G29A. Similar to 2d (control). Specifically, the binding constant for human IgG was 10 9 M ⁇ 1 or more for all ligands. Any modified C-G29A. In the neutral pH region 2d, C-G29A. The antibody binding ability was comparable to that of 2d.
  • Example 8 Modified C-G29A. Evaluation of separation behavior of antibody and host-derived impurities of 2d affinity separation matrix C-G29A / I31L. An antibody elution test was performed under the following conditions using an affinity separation matrix in which 2d was immobilized and a CHO cell culture supernatant (Biocellos) containing TNF receptor-Fc fusion protein (Etanercept). C-G29A. Prepared similarly as a control. An elution test using a 2d affinity separation matrix was also performed. The antibody recovery rate was calculated by measuring the absorbance of the eluate. Host-derived impurities (HCP) were quantified using a CHO HCP ELISA kit (Cygnus). Control C-G29A. The results using the 2d affinity separation matrix are shown in FIG. The results using the 2d affinity separation matrix are shown in FIG.
  • HCP Host-derived impurities
  • C-G29A Compared to the 2d affinity separation matrix, modified C-G29A.
  • the TNF receptor-Fc fusion protein was eluted from the affinity separation matrix prepared using 2d at high pH.
  • the amount of host-derived impurities contained in the eluted fraction was modified C-G29A.
  • the HCP content of the peak top fraction is C-G29A. 4187 ppm for the 2d affinity separation matrix, modified C-G29A. It was 3109 ppm for the 2d affinity separation matrix. This result shows that the ligand having a high elution pH in the test using IgG Sepharose of Example 1 can reduce the amount of host-derived impurities in the eluate compared to the ligand before modification.

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Abstract

L'invention fournit un ligand de protéine A dont la capacité de liaison à un anticorps diminue dans une région de pH acide, lors de la fixation sur un entraîneur et de la fabrication d'une matrice de séparation par affinité. Plus précisément, l'invention fournit une protéine qui contient une séquence d'acides aminés obtenue par substitution d'un résidu d'acides aminés hydrophobe d'une zone de liaison Fc par un autre résidu d'acides aminés hydrophobe ou un résidu d'acides aminés non chargé polaire, concernant une séquence d'acides aminés dérivée de domaines E, D, A, B ou C de la protéine A susmentionnée de SEQ ID N° 1 à 5, et qui est caractéristique en ce que sa capacité de liaison à un anticorps diminue dans une région de pH acide, en comparaison avec la protéine avant ladite substitution.
PCT/JP2016/071356 2015-07-22 2016-07-21 PROTÉINE DE LIAISON À UN ANTICORPS À CAPACITÉ DE LIAISON À UN ANTICORPS DIMINUANT DANS UNE RÉGION DE pH ACIDE Ceased WO2017014257A1 (fr)

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EP16827817.4A EP3327029A4 (fr) 2015-07-22 2016-07-21 PROTÉINE DE LIAISON À UN ANTICORPS À CAPACITÉ DE LIAISON À UN ANTICORPS DIMINUANT DANS UNE RÉGION DE pH ACIDE
JP2017529918A JP6861155B2 (ja) 2015-07-22 2016-07-21 酸性ｐＨ領域での抗体結合能が低下した抗体結合性タンパク質
CN201680042996.XA CN107849095A (zh) 2015-07-22 2016-07-21 酸性pH范围内的抗体结合能力降低的抗体结合性蛋白质
US15/876,597 US20180215795A1 (en) 2015-07-22 2018-01-22 ANTIBODY-BINDING PROTEIN HAVING REDUCED ANTIBODY-BINDING CAPACITY IN ACIDIC pH REGIONS

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